Large deletions in human BRCA1 gene and use thereof

ABSTRACT

Large deletions have been identified in the BRCA1 gene in patients. The large deletions predispose the patients to breast cancer and ovarian cancer. Thus, methods for detecting the genetic variants are provided which can be used in detecting a predisposition to cancer.

CROSS-REFERENCE TO RELATED U.S. APPLICATIONS

This application claims the benefit (under 35 U.S.C. §119(e)) of U.S.Provisional Application Ser. Nos. 60/387,132 filed on Jun. 7, 2002 and60/402,430 filed on Aug. 9, 2002, which are both incorporated herein byreference in their entirety.

TECHNICAL FIELD OF THE INVENTION

This invention generally relates to human genetics, particularly to theidentification of genetic polymorphic variations in the human BRCA1 geneand methods of using the identified genetic polymorphisms.

TECHNICAL BACKGROUND OF THE INVENTION

Breast cancer susceptibility gene 1 (BRCA1) is a tumor suppressor geneidentified on the basis of its genetic linkage to familial breastcancers. It is a 220-kilodalton nuclear phosphoprotein in normal cells.Mutations of the BRCA1 gene in humans are associated with predispositionto breast and ovarian cancers. In fact, BRCA1 and BRCA2 mutations areresponsible for the majority of familial breast cancer. Inheritedmutations in the BRCA1 and BRCA2 genes account for approximately 7-10%of all breast and ovarian cancers. Women with BRCA mutations have alifetime risk of breast cancer between 56-87%, and a lifetime risk ofovarian cancer between 27-44%. In addition, mutations in BRCA1 gene havealso been linked to various other tumors including, e.g., proliferativebreast disease (PBD), papillary serous carcinoma of the peritoneum(PSCP), and prostate cancer. Schorge, et al., J. Nat. Cancer Inst.,90:841-845 (1998); Arason, Am. J. Hum. Genet., 52:711-717 (1993);Langston, et al., New Eng. J. Med., 334: 137-142 (1996).

A large number of deleterious mutations in BRCA1 gene have beendiscovered. Genetic testing on patients to determine the presence orabsence of such deleterious mutations has proven to be an effectiveapproach in detecting predispositions to breast and ovarian cancers.Genetic testing is now commonly accepted as the most accurate method fordiagnosing hereditary breast cancer and ovarian risk.

As deleterious mutations in BRCA1 are associated with predisposition tocancers, particularly breast cancer and ovarian cancer, it is desirableto identify additional naturally existing deleterious mutations in theBRCA1 gene, which may serve as valuable diagnostic markers. One suchclass of deleterious mutations includes large deletions.

SUMMARY OF THE INVENTION

The present invention is based on the discovery of a number of largedeletions in human BRCA1 gene in patients. A detailed description of thenewly discovered deletion mutations is provided in Table 1. These largedeletions are believed to be deleterious and cause significantalterations in structure or biochemical activities in the BRCA1 geneproducts expressed from mutant BRCA1 genes. Patients with such deletionsin one of their BRCA1 genes are predisposed to, and thus have asignificantly increased likelihood of, breast cancer and/or ovariancancer. Therefore, these deletion variants are useful in genetic testingas markers for the prediction of predisposition to cancers, especiallybreast cancer and ovarian cancer, and in therapeutic applications fortreating cancers.

Accordingly, in a first aspect of the present invention, isolated BRCA1nucleic acids (genomic DNAs, corresponding mRNAs and correspondingcDNAs) are provided comprising one of the newly discovered geneticvariants summarized in Table I below.

In accordance with another aspect of the invention, isolatedpolypeptides are provided which are BRCA1 protein variants comprising atleast a portion of the amino acid sequence of a BRCA1 protein. The BRCA1protein variants are encoded by an isolated BRCA1 gene sequence of thepresent invention.

The present invention also provides a method for preparing an antibodyto a BRCA1 protein variant according to the present invention.Preferably, the antibody prepared in this method is selectivelyimmunoreactive with one or more of the newly discovered BRCA1 proteinvariants.

In accordance with another aspect of the invention, a method is providedfor genotyping BRCA1 to determine whether an individual has a geneticvariant or an amino acid variant identified in the present invention.The presence of the variants would indicate a predisposition to cancersincluding breast cancer and ovarian cancer. In accordance with thisaspect of the invention, a sample containing genomic DNA, mRNA, or cDNAof the BRCA1 gene is obtained from the individual to be tested. Thegenomic DNA, mRNA, or cDNA of the BRCA1 gene in the sample shouldinclude at least the nucleotide sequence surrounding the locus of one ormore of the above-described genetic variants such that the presence orabsence of a particular genetic variant can be determined. Any suitablemethod known in the art for genotyping can be used for determining thenucleotide(s) at a particular position in the BRCA1 gene. Alternatively,the presence or absence of one or more of the amino acid variantsdisclosed in FIG. 7, 8 or 9 can also be determined in the BRCA1 proteinin a sample isolated from a patient to be tested. The presence of thenucleotide and/or amino acid variants provided in the present inventionmay be indicative of a likelihood of a predisposition to cancers, e.g.,breast cancer and ovarian cancer.

In accordance with another aspect of the present invention, a variety ofmethods are provided for predicting a predisposition to cancer in apatient. In one embodiment these methods comprise detecting a deletionin the BRCA1 gene that can result from an unequal crossover eventbetween specific pairs of Alu sequences, wherein the presence of such adeletion would indicate a predisposition to cancer. The detection stepused in such methods can involve the analysis of BRCA1 genomic DNA, cDNAor polypeptides. Analyses of nucleic acids in these instances caninvolve amplification-based approaches or hybridization-basedapproaches. Analyses of polypeptides can involve determining whether ornot the variant BRCA1 polypeptide is truncated, or containscharacteristic epitopes that can be specifically detected with anappropriate antibody.

In another embodiment of this aspect of the present invention thesemethods comprise detecting a deletion in the BRCA1 gene that can resultfrom an unequal crossover event between specific repetitive sequences,commonly referred to as recombination breakpoints or regions, andpresented in Table 1, wherein the presence of such a deletion would alsoindicate a predisposition to cancer. As with deletions resulting fromthe unequal crossover between specific Alu repeats, the detection stepused in the methods of this embodiment can involve the analysis of BRCA1genomic DNA, cDNA or polypeptides, and anlysis of nucleic acids caninvolve amplification-based approaches.

In yet another embodiment of this aspect of the present invention thesemethods involve detecting specific sequences in BRCA1 genomic DNA orcDNA that are formed by the joining of the normally-separated sequencesthat occur on either side of the deleted region. Detection of theseindicative or characteristic nucleic acids in these instances caninvolve amplification-based approaches or hybridization-basedapproaches.

In accordance with another aspect of the invention, a detection kit isalso provided for detecting, in an individual, an elevated risk ofcancer. In a specific embodiment, the kit is used in determining apredisposition to breast cancer and ovarian cancer. The kit may include,in a partitioned carrier or confined compartment, any nucleic acidprobes or primers, or antibodies useful for detecting the BRCA1 variantsof the present invention as described above. The kit can also includeother reagents such as reverse transcriptase, DNA polymerase, buffers,nucleotides and other items that can be used in detecting the geneticvariations and/or amino acid variants according to the method of thisinvention. In addition, the kit preferably also contains instructionsfor its use.

The present invention further provides a method for identifying acompound for treating or preventing cancers associated with a BRCA1genetic variant of the present invention. The method includes screeningfor a compound capable of selectively interacting with a BRCA1 proteinvariant of the present invention.

The foregoing and other advantages and features of the invention, andthe manner in which the same are accomplished, will become more readilyapparent upon consideration of the following detailed description of theinvention taken in conjunction with the accompanying examples anddrawings, which illustrate preferred and exemplary embodiments.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1-6 show alignments of the upstream and downstream BRCA1 sequencesinvolved in the unequal crossover events that resulted in deletions 1-6,respectively. Recombination likely occurred between the regionsunderlined in the upstream and downstream sequences, to produce theobserved recombinant sequences shown (with their region of recombinationunderlined). These observed recombinant sequences were discovered ingenomic DNA isolated from human patients. The nucleotide numbers showncorrespond to the reference sequence provided by GenBank AccessionNumber L78833.1 (Smith et al., Genome Res. 6:1029-1049 (1996)).

FIGS. 7, 8, and 9 depict the effects or consequences of the newlydiscovered large deletions on the gene products of BRCA1 genes bearingsuch mutations. In particular, FIG. 7 illustrates the effects of newlydiscovered deletion Nos. 1, 2, 3, and 4, which effectively remove exons16 and 17 from the BRCA1 gene transcript (mRNA), thereby removing the133 codons encoding amino acid residues E1559-T1691. Although thesemutations all result in a shortened mRNA transcript lacking exons 16 and17 and a shortened mutant BRCA1 protein, they do not disrupt the openreading frame of the remaining transcript.

FIG. 8 shows the effects of newly discovered Deletion No. 5, whicheffectively removes exons 15 and 16 from the BRCA1 gene transcript(mRNA), thereby removing the third position nucleotide from codon R1495and the following 167 codons encoding amino acid residues S1496-F1661.Removal of the third position nucleotide of codon R1495 serves todisrupt the downstream open reading frame, resulting in a frame shiftthat is maintained until an ochre stop codon is encountered fourteencodons into exon 17. As a result of the frame-shift created by theremoval of exons 15 and 16 from the spliced gene transcript, a novel13-amino acid sequence encoded by the frame-shifted exon 17 (SEQ IDNO:13) is append onto R1495 of the translated BRCA1 polypeptide, and theoverall length of the resulting BRCA1 protein is shortened from 1863 to1507 amino acid residues.

FIG. 9 depicts the effects of newly discovered deletion No. 6, whicheffectively removes exons 14 through 20 from the BRCA1 gene transcript(mRNA), thereby removing the second and third position nucleotides fromcodon A1452 and the following 306 codons encoding amino acid residuesV1453-K1758. Removal of second and third position nucleotides from codonA1453 serves to disrupt the downstream open reading frame, resulting ina translational frame shift that is maintained through the codonsencoded by exons 21, 22, 23 and 24, until a UGA stop codon isencountered 7 codons into exon 24. As a result of the frame-shiftcreated by the removal of exons 14 through 20 from the spliced genetranscript, a novel 69-amino acid sequence encoded by the frame-shiftedexons 21, 22, 23, and 24 (SEQ ID NO:14) is append onto K1452 of thetranslated BRCA1 polypeptide, and the overall length of the resultingBRCA1 protein is shortened from 1863 to 1521 amino acid residues.

Note: For FIGS. 1-6, the BRCA1 genomic DNA nucleotide or basepairnumbers correspond to the reference sequence provided by GenBankAccession Number L78833.1. For FIGS. 7, 8, and 9, the BRCA1 cDNAnucleotide and amino acid numbers correspond to the reference sequenceprovided by GenBank Accession No. U14680.1.

DETAILED DESCRIPTION OF THE INVENTION

1. Definitions

The terms “genetic variant,” “mutation,” and “nucleotide variant” areused herein interchangeably to refer to changes or alterations to areference BRCA1 gene sequence at a particular locus, including, but notlimited to, nucleotide base deletions, insertions, inversions, andsubstitutions in the coding and noncoding regions. Deletions may be of asingle nucleotide, a portion or a region of the nucleotide sequence ofthe gene, or of the entire gene sequence. Insertions may be of one ormore nucleotides. The genetic variants may occur in transcriptionalregulatory regions, untranslated regions of mRNA, exons, introns, orexon/intron junctions. The genetic variants may or may not result instop codons, frame shifts, deletion of amino acids, altered amino acidsequence, or altered protein expression level. The mutations or geneticvariants can be somatic, i.e., occur only in certain tissues of the bodyand are not inherited in the germline, or germline mutations, i.e.,inherited mutations found in all tissues.

“Genetic polymorphism” as used herein refers to the phenomena that twoor more genetic variants in a particular locus of a gene are found in apopulation.

The term “allele” or “gene allele” is used herein to refer generally toa naturally occurring gene having the reference sequence or a genecontaining a specific genetic variant.

As used herein, the term “BRCA1 nucleic acid” means a nucleic acidmolecule the nucleotide sequence of which is found uniquely in a BRCA1gene or a substantially equivalent form thereof. That is, the nucleotidesequence of a “BRCA1 nucleic acid” can be a full-length sequence of, ora portion found in, either BRCA1 genomic DNA or mRNA/cDNA, eitherwild-type or naturally existing variant BRCA1 gene, or an artificialnucleotide sequence encoding a wild-type BRCA1 protein or naturallyexisting polymorphic variant BRCA1 protein.

The term “BRCA1 nucleic acid variant” refers to a naturally existingBRCA1 nucleic acid.

As used herein, the term “amino acid variant” refers to amino acidchanges to a reference BRCA1 protein sequence resulting from nucleotidevariants or mutations to the reference gene encoding the reference BRCA1protein. The term “amino acid variant” is intended to encompass not onlysingle amino acid substitutions, but also amino acid deletions,insertions, and other significant changes of amino acid sequence in aBRCA1 protein.

The term “BRCA1 protein variant” is used herein relative to a referenceBRCA1 protein to mean a BRCA1 protein found in a population that is thecoding product of a BRCA1 gene allele containing genetic variants suchas single nucleotide substitutions, insertions, deletions, and DNArearrangements, which lead to alterations in the protein sequence of theprotein variant.

The term “locus” refers to a specific position or site in a nucleotidesequence of a gene, or amino acid sequence of a protein. Thus, there maybe one or more contiguous nucleotides in a particular gene locus, or oneor more amino acids at a particular locus in a polypeptide. Moreover,“locus” may also be used to refer to a particular position in a genesequence where one or more nucleotides have been deleted, inserted, orinverted.

The terms “polypeptide,” “protein,” and “peptide” are used hereininterchangeably to refer to amino acid chains in which the amino acidresidues are linked by peptide bonds or modified peptide bonds. Theamino acid chains can be of any length of greater than two amino acids.Unless otherwise specified, the terms “polypeptide,” “protein,” and“peptide” also encompass various modified forms thereof. Such modifiedforms may be naturally occurring modified forms or chemically modifiedforms.

Examples of modified forms include, but are not limited to, glycosylatedforms, phosphorylated forms, myristoylated forms, palmitoylated forms,ribosylated forms, acetylated forms, etc. Modifications also includeintra-molecular crosslinking and covalent attachment of various moietiessuch as lipids, flavin, biotin, polyethylene glycol or derivativesthereof, etc. In addition, modifications may also include cyclization,branching and cross-linking. Further, amino acids other than theconventional twenty amino acids encoded by genes may also be included ina polypeptide.

The terms “primer,” “probe,” and “oligonucleotide” may be used hereininterchangeably to refer to a relatively short nucleic acid fragment orsequence. They can be DNA, RNA, or a hybrid thereof, or chemicallymodified analogs or derivatives thereof. Typically, they aresingle-stranded. However, they can also be double-stranded having twocomplementing strands that can be separated apart by denaturation.Normally, they have a length of from about 8 nucleotides to about 200nucleotides, preferably from about 12 nucleotides to about 100nucleotides, and more preferably about 18 to about 50 nucleotides. Theycan be labeled with detectable markers or modified in any conventionalmanners for various molecular biological applications.

The term “isolated,” when used in reference to nucleic acids (whichinclude gene sequences or fragments) of this invention, is intended tomean that a nucleic acid molecule is present in a form other than foundin nature in its original environment with respect to its associationwith other molecules. For example, since a naturally existing chromosomeincludes a long nucleic acid sequence, an “isolated nucleic acid” asused herein means a nucleic acid molecule having only a portion of thenucleic acid sequence in the chromosome but not one or more otherportions present on the same chromosome. Thus, for example, an isolatedgene typically includes no more than 25 kb of naturally occurringnucleic acid sequence which immediately flanks the gene in the naturallyexisting chromosome or genomic DNA. However, it is noted that an“isolated nucleic acid” as used herein is distinct from a clone in aconventional library such as genomic DNA library and cDNA library inthat the clones in a library are still in admixture with almost all theother nucleic acids in a chromosome or a cell. An isolated nucleic acidcan be in a vector.

The term “isolated nucleic acid” embraces “purified nucleic acid” whichmeans a specified nucleic acid is in a substantially homogenouspreparation of nucleic acid substantially free of other cellularcomponents, other nucleic acids, viral materials, or culture medium, orchemical precursors or by-products associated with chemical reactionsfor chemical synthesis of nucleic acids. Typically, a “purified nucleicacid” can be obtained by standard nucleic acid purification methods. Ina purified nucleic acid, preferably the specified nucleic acid moleculeconstitutes at least 15 percent of the total nucleic acids in thepreparation. The term “purified nucleic acid” also means nucleic acidsprepared from a recombinant host cell (in which the nucleic acids havebeen recombinantly amplified and/or expressed), or chemicallysynthesized nucleic acids.

The term “isolated nucleic acid” also encompasses a “recombinant nucleicacid” which is used herein to mean a hybrid nucleic acid produced byrecombinant DNA technology having the specified nucleic acid moleculecovalently linked to one or more nucleic acid molecules that are not thenucleic acids naturally flanking the specified nucleic acid. Typically,such nucleic acid molecules flanking the specified nucleic acid are nomore than 50 kb. In addition, the specified nucleic acid may have anucleotide sequence that is identical to a naturally occurring nucleicacid, or a modified form, or mutant form thereof having one or moremutations such as nucleotide substitution, deletion/insertion,inversion, and the like.

In addition, “isolated nucleic acid” further includes a chemicallysynthesized nucleic acid having a naturally occurring nucleotidesequence or an artificially modified form thereof (e.g., dideoxy forms).

The term “isolated polypeptide” as used herein means a polypeptidemolecule is present in a form other than found in nature in its originalenvironment with respect to its association with other molecules. Theterm “isolated polypeptide” encompasses a “purified polypeptide” whichis used herein to mean that a specified polypeptide is in asubstantially homogenous preparation, substantially free of othercellular components, other polypeptides, viral materials, or culturemedium, or when the polypeptide is chemically synthesized, substantiallyfree of chemical precursors or by-products associated with the chemicalsynthesis. For a purified polypeptide, preferably the specifiedpolypeptide molecule constitutes at least 15 percent of the totalpolypeptide in the preparation. A “purified polypeptide” can be obtainedfrom natural or recombinant host cells by standard purificationtechniques, or by chemical synthesis.

The term “isolated polypeptide” also encompasses a “recombinantpolypeptide,” which is used herein to mean a hybrid polypeptide producedby recombinant DNA technology or chemical synthesis having a specifiedpolypeptide molecule covalently linked to one or more polypeptidemolecules which do not naturally link to the specified polypeptide.

As used herein, “haplotype” is a combination of genetic (nucleotide)variants in a region of an mRNA or a genomic DNA on a chromosome foundin an individual. Thus, a haplotype includes a number of geneticallylinked polymorphic variants that are typically inherited together as aunit.

The term “reference sequence” refers to a polynucleotide or polypeptidesequence known in the art, including those disclosed in publiclyaccessible databases (e.g., GenBank), or a newly identified genesequence, used simply as a reference with respect to the variantsprovided in the present invention. The nucleotide or amino acid sequencein a reference sequence is contrasted to the alleles disclosed in thepresent invention having newly discovered nucleotide or amino acidvariants.

The terms “crossing-over” and “crossover,” are used interchangeablyherein, to refer to the reciprocal exchange of material betweenchromosome homologs—by breakage and reunion—that occurs during meiosisand is responsible for genetic recombination. The term “unequalcrossover,” as used herein, refers to a crossover event occurringbetween homologous sequences in paired chromosome homologs that are notperfectly aligned, or, more generally, describes a recombination eventin which the two recombining sites lie at nonidentical locations in thetwo parental DNA molecules. The products of an unequal crossover are twochromosomes, or more generally two progeny DNA molecules, one of whichbears a deletion, and the other of which bears a duplication of thenucleotide sequence residing between the mispaired homologous sequencesor recombining sites.

2. Nucleotide and Amino Acid Variants

Smith and coworkers described the complete genomic sequence of a 117kilobase region of human DNA containing the BRCA1 gene, and depositedthe nucleotide sequence of the genomic DNA in the GenBank under theAccession Number L78833.1 (Smith et al., Genome Res., 6:1029-1049(1996)). This nucleotide sequence (referred to as L78833.1) is usedherein as a reference sequence for identifying the polymorphic positionsof the large deletions of the present invention and the upstream anddownstream sequences that were likely involved in the unequal crossoverevents that yielded Deletion Nos. 1-6. The complete coding sequencecorresponding to the mRNA transcribed from the BRCA1 gene, and the aminoacid sequence encoded therein, were deposited in the GenBank underAccession Number U14680.1. These sequences (cDNA and amino acid) areused as reference sequences for identifying the effects or consequencesof the large deletions at the level of the gene transcript (mRNA), cDNAand encoded protein.

In accordance with the present invention, analysis of the nucleotidesequence of genomic DNA corresponding to the BRCA1 genes of specifichuman patients has led to the discovery of a number of mutant BRCA1alleles that exhibit large deletions relative to the reference sequenceprovided by GenBank Accession No. L78833.1. Specifically, six differentgenetic variants exhibiting large deletions have been discovered. Thesesix different genetic variants, and the effects or consequences theyhave on the gene products expressed from the BRCA1 alleles that bearthem, are summarized in Table 1. Of these six different genetic variantscorresponding to six different large deletions of nucleotide sequencewithin the BRCA1 gene, four result in the deletion of exons 16 and 17,one results in deletion of exons 15 and 16, and one results in deletionof exons 14 through 20, in the mRNAs transcribed from the variantalleles.

TABLE I GENETIC VARIANTS OF THE BRCA1 GENE All numeric designation ofnucleotides conform to the sequence in Smith et al., Genome Res.,6:1029–1049 (1996) and GenBank Accession Number L78833.1 RecombinationBreakpoint Size of Deletion 5′ Region 3′ Region Deletion Exons No. (ntpositions) (nt positions) (bp) Removed Consequences of Deletion 156,960–56,998 63,296–63,334 6,337 16 & 17 Removal of residuesE1559-T1691 2 54,960–54,965 62,143–62,147 7,183 16 & 17 Removal ofresidues E1559-T1691 3 55,893–55,932 62,049–62,088 6,157 16 & 17 Removalof residues E1559-T1691 4 56,090–56,095 61,838–61,843 5,749 16 & 17Removal of residues E1559-T1691 5 53,030–53,075 58,659–58,704 5,629 15 &16 Addition of a novel 13-residue carboxyl-terminus onto R1495 650,524–50,577 76,977–77,031 26,454 14–20 Addition of a novel 69-residuecarboxyl-terminus onto K1452

In further accordance with the present invention, the large deletionsdescribed in Table 1 were found in patients at high risk of developingbreast cancer. Nucleotide sequences obtained from these individualsindicate that all six of these large deletions involved the joining of aparticular sequence in a more 5′ region of the BRCA1 gene (an upstreamsequence), to a similar sequence in a more 3′ region of the BRCA1 gene(a downstream sequence) to create a recombined or joined sequence thatspans the deletion locus. Further analysis has shown that all upstreamsequences, and all downstream sequences reside within identified Alurepeats (Smith et al, Genome Res., 6:1029-1049 (1996)). Consequently,the observed mutations most likely arose from an unequal crossover eventoccurring between misaligned Alu sequences in the BRCA1 genes of pairedhomologous chromosomes. The specific sequences of the upstream anddownstream loci involved in these six unequal crossover events, alongwith the specific joined or recombined sequences resulting from theseunequal crossover events (the “deletion loci”), which have been observedin the genomic DNA of specific individuals, are shown in FIGS. 1-6. Theconsequences of each of the large deletions observed in mutant BRCA1genomic DNAs (as depicted in FIGS. 1-6) on the nucleotide sequence ofthe mRNA transcript transcribed therefrom (or on the correspondingcDNA), as well as on the amino acid sequence of the encoded protein, areshown in FIGS. 7-9.

The breakpoint regions (upstream and downstream loci) believed to beresponsible for the unequal crossover that resulted in Deletion No. 1,and the resulting recombined nucleotide sequence discovered in humanpatients are shown underlined in FIG. 1. As indicated in Table 1, the 5′recombination breakpoint resides between nucleotides 56,960 and 56,998(underlined in the upstream sequence) and the 3′ recombinationbreakpoint resides between nucleotides 63,296 and 63,334 (underlined inthe downstream sequence). Recombination between the upstream anddownstream breakpoint regions has resulted in the deletion of 6,337basepairs of the BRCA1 gene and has produced a novel BRCA1 gene sequencecomprising the junction sequence provided by SEQ ID NO:1. The resultingrecombined genomic DNA sequence, which when transcribed directs theexpression of mutant mRNAs lacking exons 16 and 17 (FIG. 7), was foundin three individuals.

The loci (breakpoint regions) believed to be responsible for the unequalcrossover that resulted in Deletion No. 2, and the resulting recombinednucleotide sequence discovered in human patients are shown underlined inFIG. 2. As indicated in Table 1, the 5′ recombination breakpoint residesbetween nucleotides 54,960 and 54,965 (underlined in the upstreamsequence) and the 3′ recombination breakpoint resides betweennucleotides 62,143 and 62,147 (underlined in the downstream sequence).Recombination between the upstream and downstream breakpoint regions hasresulted in the deletion of 7,183 basepairs of the BRCA1 gene and hasproduced a novel BRCA1 gene sequence comprising the junction sequenceprovided by SEQ ID NO:2. The resulting recombined genomic DNA sequence,which when transcribed also directs the expression of mutant mRNAslacking exons 16 and 17 (FIG. 7), was identified in one individual.

The loci (breakpoint regions) believed to be responsible for the unequalcrossover that resulted in Deletion No. 3, and the resulting recombinednucleotide sequence discovered in human patients are shown underlined inFIG. 3. As indicated in Table 1, the 5′ recombination breakpoint residesbetween nucleotides 55,893 and 55,932 (underlined in the upstreamsequence) and the 3′ recombination breakpoint resides betweennucleotides 62,048 and 62,087 (underlined in the downstream sequence).Recombination between the upstream and downstream breakpoint regions hasresulted in the deletion of 6,157 basepairs of the BRCA1 gene and hasproduced a novel BRCA1 gene sequence comprising the junction sequenceprovided by SEQ ID NO:3. The resulting recombined genomic DNA sequence,which when transcribed also directs the expression of mutant mRNAslacking exons 16 and 17 (FIG. 7), was characterized in one individual.

The loci (breakpoint regions) believed to be responsible for the unequalcrossover that resulted in Deletion No. 4, and the resulting recombinednucleotide sequence discovered in human patients are shown underlined inFIG. 4. As indicated in Table 1, the 5′ recombination breakpoint residesbetween nucleotides 56,090 and 56,095 (underlined in the upstreamsequence) and the 3′ recombination breakpoint resides betweennucleotides 61,838 and 61,843 (underlined in the downstream sequence).Recombination between the upstream and downstream breakpoint regions hasresulted in the deletion of 5,749 basepairs of the BRCA1 gene and hasproduced a novel BRCA1 gene sequence comprising the junction sequenceprovided by SEQ ID NO:4. The resulting recombined genomic DNA sequence,which when transcribed also directs the expression of mutant mRNAslacking exons 16 and 17 (FIG. 7), was found in one individual.

The loci (breakpoint regions) believed to be responsible for the unequalcrossover that resulted in Deletion No. 5, and the resulting recombinednucleotide sequence discovered in human patients are shown underlined inFIG. 5. As indicated in Table 1, the 5′ recombination breakpoint residesbetween nucleotides 53,030 and 53,075 (underlined in the upstreamsequence) and the 3′ recombination breakpoint resides betweennucleotides 58,659 and 58,704 (underlined in the downstream sequence).Recombination between the upstream and downstream breakpoint regions hasresulted in the deletion of 5,629 basepairs of the BRCA1 gene and hasproduced a novel BRCA1 gene sequence comprising the junction sequenceprovided by SEQ ID NO:5. The resulting recombined genomic DNA sequence,which when transcribed also directs the expression of mutant mRNAslacking exons 15 and 16 (FIG. 8), was identified in one individual.

The loci (breakpoint regions) believed to be responsible for the unequalcrossover that resulted in Deletion No. 6, and the resulting recombinednucleotide sequence discovered in human patients are shown underlined inFIG. 6. As indicated in Table 1, the 5′ recombination breakpoint residesbetween nucleotides 50,524 and 50,577 (underlined in the upstreamsequence) and the 3′ recombination breakpoint resides betweennucleotides 76,977 and 77,031 (underlined in the downstream sequence).Recombination between the upstream and downstream breakpoint regions hasresulted in the deletion of 26,454 basepairs of the BRCA1 gene and hasproduced a novel BRCA1 gene sequence comprising the junction sequenceprovided by SEQ ID NO:6. The resulting recombined genomic DNA sequence,which when transcribed also directs the expression of mutant mRNAslacking exons 14 through 20 (FIG. 9), has now been characterized infourteen individuals.

The consequences of Deletions 1-6 on the gene products encoded by theBRCA1 alleles bearing these mutations are depicted in FIGS. 7-9. Asmentioned above, Deletion Nos. 1, 2, 3, and 4 all produce mutant allelesof the BRCA1 gene that, when transcribed, direct the expression of mRNAslacking exons 16 and 17 (FIG. 7). Such mRNAs, and cDNAs prepared fromthem, lack the codons encoding amino acid residues E1559-T1691, and arecharacterized by the novel junction sequence comprising SEQ ID NO:7,which spans the deleted codons. Despite the omission of the 133 codonsencoded by exons 16 and 17, the open reading frame of the remainingnucleotides is not disrupted (i.e., ntG4675 carries over to ntA5075 andntT5076, so that aaD1692 is conserved). Consequently, the mRNAstranscribed from the mutant alleles characterized by Deletion Nos. 1-4,direct the translation of a mutant BRCA1 protein comprised of 1,730amino acid residues, instead of the normal 1,863. These shorter mutantBRCA1 proteins are characterized by the amino acid sequence created bythe juxtaposition of the codon encoding L1558 with the codon encodingD1692, and comprising SEQ ID NO:10.

In contrast, Deletion No. 5 produces a mutant allele of the BRCA1 genethat, when transcribed, directs the expression of mRNA lacking exons 15and 16 (FIG. 8). Such mRNA, and the cDNA prepared from it, lacks thecodons encoding amino acid residues S1496-F1662, as well as the thirdposition nucleotide from codon R1495, and is characterized by the noveljunction sequence comprising SEQ ID NO:8, which spans the region of thedeleted codons. Unlike with Deletions Nos. 1-4, mRNA transcribed frommutant BRCA1 alleles encompassing Deletion No. 5 directs a translationalframe shift downstream of the junction between nucleotides encoded byexons 14 and 17. Translation in the shifted frame is maintained until anochre stop codon is encountered fourteen codons into exon 17. As aresult of the frame-shift created by the omission of exons 15 and 16, anovel 13-amino acid sequence (SEQ ID NO:13), encoded by codons withinthe frame-shifted exon 17, is appended onto R1495 of the translatedmutant BRCA1 polypeptide, and the overall length of the BRCA1 protein isshortened from 1863 to 1507 amino acid residues. Consequently, thesemutant BRCA1 proteins are characterized by their shortened length, theirnovel carboxy-termini, and by the unique amino acid sequence created bythe splicing of exons 14 and 17, which comprises SEQ ID NO:11.

Deletion No. 6 produces a mutant allele of the BRCA1 gene that, whentranscribed, directs the expression of mRNA lacking exons 14 through 20(FIG. 9). Such mRNA, and the cDNA prepared from it, lacks the codonsencoding amino acid residues V1453-K1758, as well as the second andthird position nucleotides from codon A1453, and is characterized by thenovel junction sequence comprising SEQ ID NO:9, which spans the deletedcodons. Like Deletion No. 5, Deletion No. 6 directs a translationalframe shift downstream of the junction between nucleotides encoded byexons 13 and 21. Translation in the shifted frame is maintained throughthe codons encoded by exons 21, 22, 23 and 24, until a UGA stop isencountered 7 codons into exon 24. As a result of the frame-shiftcreated by the omission of exons 14 through 20, a novel 69-amino acidsequence (SEQ ID NO:14) is appended onto K1452 of the translated mutantBRCA1 polypeptide, and the overall length of the BRCA1 protein isshortened from 1863 to 1521 amino acid residues. Consequently, thesemutant BRCA1 proteins are also characterized by their shortened length,their novel carboxy-termini, and by the unique amino acid sequencecreated by the splicing of exons 13 and 21, which comprises SEQ IDNO:12.

As shown in the Figures, and described above, the genetic variantsaccording to the present invention are expected to cause significantchanges in the structure and biological activity of the BRCA1 proteinthey encode. Individuals who inherit such genetic variants (largedeletion mutations) are predisposed to cancers, particularly breastcancer and ovarian cancer.

3. BRCA1 Nucleic Acids

In a first aspect of the present invention, isolated nucleic acids areprovided comprising a nucleotide sequence of a BRCA1 nucleic acidvariant identified in accordance with the present invention. Thenucleotide sequence is at least 12, 13, 14, 15, 17, 18, 19, 20, 25, 30,or 35 contiguous nucleotides spanning the deletion locus in one of themutant BRCA1 genomic DNAs having one of the Deletion Nos. 1-6, or thedeletion locus in one of the mutant BRCA1 mRNAs, or cDNAs preparedtherefrom, expressed from the mutant BRCA1 genomic DNAs having one ofthe Deletion Nos. 1-6. The nucleic acid molecules can be in a form ofDNA, RNA, or a chimeric or hybrid thereof, and can be in any physicalstructures including a single-strand or double-strand or in the form ofa triple helix.

In one embodiment, the isolated nucleic acids have a sequence selectedfrom the group consisting of SEQ ID NOs:1-9 or 15-82, and complementsthereof. Specifically, SEQ ID NOs:1 and 15-17 are mutant sequences seenin the BRCA1 genomic DNA variant that resulted from Deletion No. 1. SEQID NOs:2 and 18-23 are mutant sequences seen in the BRCA1 genomic DNAvariant that resulted from Deletion No. 2. SEQ ID NOs:3 and 24-27 aremutant sequences seen in the BRCA1 genomic DNA variant that resultedfrom Deletion No. 3. SEQ ID NOs:4 and 28-32 are mutant sequences seen inthe BRCA1 genomic DNA variant that resulted from Deletion No. 4. SEQ IDNOs:5 and 33-36 are mutant BRCA1 genomic sequences that resulted fromDeletion No. 5. And, SEQ ID NOs:6 and 37-41 are mutant BRCA1 genomicsequences that resulted from Deletion No. 6.

In addition, SEQ ID NOs:7 and 42-52 are mutant BRCA1 cDNA sequences thatspan the cDNA deletion locus that results from Deletion Nos. 1, 2, 3,and 4. SEQ ID NOs:45-47 are portions of the antisense strand sequence ofthe mutant BRCA1 cDNA resulted from Deletion Nos. 1, 2, 3, and 4, whileSEQ ID NOs:7, 42-44, and 48-52 are portions of the sense strand thatalso span the cDNA deletion locus resulted from Deletion Nos. 1, 2, 3,and 4.

SEQ ID NOs:8, 53-57, and 64-66 are portions of the sense strand ofmutant BRCA1 cDNA sequences that span the cDNA deletion locus resultedfrom Deletion No. 5, while SEQ ID NOs:58-63 are portions of theantisense strand sequence of the mutant BRCA1 cDNA resulted fromDeletion No. 5.

SEQ ID NOs:9, 67-74, and 64-66 are portions of the sense strand ofmutant BRCA1 cDNA sequences that span the cDNA deletion locus resultedfrom Deletion No. 6, while SEQ ID NOs:75-79 are portions of theantisense strand sequence of the mutant BRCA1 cDNA resulted fromDeletion No. 6.

SEQ ID NOs:80 represents the portion of the cDNA encoding the novelcarboxyl-terminal tail of the mutant BRCA1 polypeptide resulting fromDeletion No. 5. SEQ ID NO:81 represents the cDNA encompassing thejunction of the original reading frame and the novel frame-shiftedreading frame that results from the juxtaposition of exons 13 and 21seen in the mutant BRCA1 mRNA resulting from Deletion No. 6, while SEQID NO:82 represents the portion of the cDNA encoding the novelcarboxyl-terminal tail of the mutant BRCA1 polypeptide resulting fromDeletion No. 6.

In a specific embodiment, the isolated nucleic acids of the presentinvention are isolated BRCA1 nucleic acid having a sequence according toone of SEQ ID NOs:1-9 or 15-82, or complements thereof. Preferably, theisolated BRCA1 nucleic acids are isolated BRCA1 nucleic acid variantsthat are mutant BRCA1 genomic DNAs having one of the Deletion Nos. 1-6,or those mutant BRCA1 mRNAs derived from the mutant BRCA1 genomic DNAs,having one of the Deletion Nos. 1-6, or cDNAs derived from such mRNAs.The BRCA1 genomic DNAs, cDNAs and mRNAs can have a full-length sequence(i.e., including the entire coding regions and, in the case of genomicDNAs, optionally introns, promoter, and other regulatory sequences) orpartial sequence (i.e., a portion of the full-length sequence).

In one embodiment, an isolated BRCA1 nucleic acid is an oligonucleotide,primer or probe comprising a contiguous span of the nucleotide sequenceof a mutant BRCA1 sequence (either genomic DNA or cDNA or mRNA sequence)provided in accordance with the present invention and spanning a cDNAdeletion locus resulted from Deletion Nos. 1, 2, 3, 4, 5 or 6. Theoligonucleotide, primer or probe contains at least 12, preferably fromabout 15, 18, 20, 22, 25, 30, 40 to about 50, 60, 70, 80, 90, or 100,and more preferably from about 30 to about 50 nucleotides. In oneembodiment, the oligonucleotides, primers and probes are specific to aBRCA1 nucleic acid variant of the present invention. That is, theyselectively hybridize, under stringent conditions generally recognizedin the art, to a BRCA1 nucleic acid variant of the present invention,but do not substantially hybridize to a reference BRCA1 nucleic acidsequence under stringent conditions. Such oligonucleotides will beuseful in hybridization-based methods, or alternativelyamplification-based methods, for detecting the nucleotide variants ofthe present invention as described in detail below. A skilled artisanwould recognize various stringent conditions that enable theoligonucleotides of the present invention to differentiate between areference BRCA1 gene sequence and an isolated BRCA1 nucleic acid variantof the present invention. For example, the hybridization can beconducted overnight in a solution containing 50% formamide, 5×SSC,pH7.6, 5× Denhardt's solution, 10% dextran sulfate, and 20 microgram/mldenatured, sheared salmon sperm DNA. The hybridization filters can bewashed in 0.1×SSC at about 65° C.

The oligonucleotide primers or probes of the present invention can havea detectable marker selected from, e.g., radioisotopes, fluorescentcompounds, enzymes, or enzyme co-factors operably linked to theoligonucleotide. The primers, probes and oligonucleotide sequences ofthe present invention are useful in genotyping and haplotyping as willbe apparent from the description below.

In another specific embodiment, BRCA1 nucleic acids are provided having100, 200, 300, 400 or 500 nucleotides or basepairs, which contain theBRCA1 variant nucleotide or basepair sequences provided by SEQ IDNOs:1-9 or 15-82, and/or the complements thereof. Such nucleic acids canbe DNA or RNA, and single-stranded or double-stranded.

It should be understood that any nucleic acid molecules containing asequence according to one of SEQ ID NOs: 1-9 or 15-82 fall within thescope of this invention. For example, a hybrid nucleic acid molecule maybe provided having a sequence according to one of SEQ ID NOs: 1-9 or15-82 operably linked to a non-BRCA1 sequence such that the hybridnucleic acid encodes a hybrid protein having a mutant BRCA1 peptidesequence. In another embodiment, the present invention provides a vectorconstruct containing one of the nucleic acid molecules of the presentinvention. As will be apparent to skilled artisans, the vector may beemployed to amplify a nucleic acid molecule of the present inventionthat is contained in the vector construct. Alternatively, the vectorconstruct may be used in expressing a polypeptide encoded by a nucleicacid molecule of the present invention that is contained in the vectorconstruct. Generally, the vector construct may include a promoteroperably linked to an isolated nucleic acid molecule (including afull-length sequence or a fragment thereof in the 5′ to 3′ direction orin the reverse direction for the purpose of producing antisense nucleicacids), an origin of DNA replication for the replication of the vectorsin host cells and a replication origin for the amplification of thevectors in, e.g., E. coli, and selection marker(s) for selecting andmaintaining only those host cells harboring the vectors. Additionally,the vectors preferably also contain inducible elements, which functionto control the expression of the isolated gene sequence. Otherregulatory sequences such as transcriptional termination sequences andtranslation regulation sequences (e.g., Shine-Dalgarno sequence) canalso be included. An epitope tag coding sequence for detection and/orpurification of the encoded polypeptide can also be incorporated intothe vector construct. Examples of useful epitope tags include, but arenot limited to, influenza virus hemagglutinin (HA), Simian Virus 5 (V5),polyhistidine (6×His), c-myc, lacZ, GST, and the like. Proteins withpolyhistidine tags can be easily detected and/or purified with Niaffinity columns, while specific antibodies to many epitope tags aregenerally commercially available. The vector construct can be introducedinto the host cells or organisms by any techniques known in the art,e.g., by direct DNA transformation, microinjection, electroporation,viral infection, lipofection, biolystics (gene gun), and the like. Thevector construct can be maintained in host cells in an extrachromosomalstate, i.e., as self-replicating plasmids or viruses. Alternatively, thevector construct can be integrated into chromosomes of the host cells byconventional techniques such as selection of stable cell lines orsite-specific recombination. The vector construct can be designed to besuitable for expression in various host cells, including but not limitedto bacteria, yeast cells, plant cells, insect cells, and mammalian andhuman cells. A skilled artisan will recognize that the designs of thevectors can vary with the host used.

In another embodiment, a BRCA1 nucleic acid of the present invention isincorporated in a microchip or microarray, or other similar structures.The microarray will allow rapid genotyping and/or haplotyping in a largescale. As is known in the art, in microchips, a large number ofdifferent nucleic acids are attached or immobilized in an array on asolid support, e.g., a silicon chip or glass slide. Target nucleic acidsequences to be analyzed can be contacted with the immobilized nucleicacids on the microchip. See Lipshutz et al., Biotechniques, 19:442-447(1995); Chee et al., Science, 274:610-614 (1996); Kozal et al., Nat.Med. 2:753-759 (1996); Hacia et al., Nat. Genet., 14:441-447 (1996);Saiki et al., Proc. Natl. Acad. Sci. USA, 86:6230-6234 (1989); Gingeraset al., Genome Res., 8:435-448 (1998). The microchip technologiescombined with computerized analysis tools allow large-scale highthroughput screening. See, e.g., U.S. Pat. No. 5,925,525 to Fodor et al;Wilgenbus et al., J. Mol. Med., 77:761-786 (1999); Graber et al., Curr.Opin. Biotechnol., 9:14-18 (1998); Hacia et al., Nat. Genet., 14:441-447(1996); Shoemaker et al., Nat. Genet., 14:450-456 (1996); DeRisi et al.,Nat. Genet., 14:457-460 (1996); Chee et al., Nat. Genet., 14:610-614(1996); Lockhart et al., Nat. Genet., 14:675-680 (1996); Drobyshev etal., Gene, 188:45-52 (1997).

In a preferred embodiment, a microarray is provided comprising aplurality of the nucleic acids of the present invention such that thenucleotide identity at each of the genetic variant sites disclosed inTable I can be determined in one single microarray.

4. BRCA1 Polypeptides

The present invention also provides isolated polypeptides having a novelamino acid sequence of a BRCA1 protein variant identified in accordancewith the present invention. The amino acid sequence is a contiguoussequence of at least 3, 4, 5, 6, 7, 8, 9, 10, 12, or 13 amino acidsspanning the deletion locus resulted from Deletion Nos. 1, 2, 3, 4, 5,or 6. In addition, the amino acid sequence can also be a contiguoussequence of at least 3, 4, 5, 6, 7, 8, 9, 10, 12, or 13 amino acidswithin the carboxyl-terminal sequence of 13 amino acids of the BRCA1protein variant resulting from Deletion No. 5. Alternatively, the aminoacid sequence can be a contiguous sequence of at least 5, 6, 7, 8, 9,10, 12, 14, 16, 18, 20, 25, 30, 35, 40 45, 50, 55, 60, 65, or 69 aminoacids within the carboxyl-terminal sequence of 69 amino acids of theBRCA1 protein variant resulting from Deletion No. 6.

In one embodiment, the isolated polypeptides of the present inventioncomprise an amino acid sequence according to one of SEQ ID NOs:10-14 or83-93. The isolated polypeptide of the present invention can have atleast 7, 8, 9, or more amino acids in length, preferably 10 or more,more preferably 25 or more, and even more preferably 50 or more aminoacids.

The isolated polypeptides of the present invention comprising an aminoacid sequence according to one of SEQ ID NOs:10 and 83-87 representnovel amino acid sequence fragments of the BRCA1 protein variantresulting from Deletion Nos. 1, 2, 3 or 4. As illustrated in FIG. 7, anddescribed above, these polypeptides correspond to codon sequences inmutant mRNAs created by the direct splicing of exon 15 to exon 18—mutantmRNAs that are transcribed from mutant alleles of the BRCA1 gene bearingeither Deletion No. 1, 2, 3, or 4—and all contain certain amino acidresidues encoded by codons representing both exons. Hence, the isolatedpolypeptides of the present invention comprising an amino acid sequenceaccording to one of SEQ ID NOs:10-14 or 83-93 all represent noveljunction polypeptides in which amino acid residues encoded by exon 15are joined with amino acid residues encoded by exon 18.

Similarly, the isolated polypeptides of the present invention comprisingan amino acid sequence according to one of SEQ ID NOs:11, 88, and 89represent novel amino acid sequence fragments of the BRCA1 proteinvariant resulting from Deletion No. 5. As shown in FIG. 8, and describedabove, these polypeptides correspond to codon sequences in mutant mRNAscreated by the direct splicing of exon 14 to exon 17—mutant mRNAs thatare transcribed from mutant alleles of the BRCA1 gene bearing DeletionNo. 5—and all contain certain amino acid residues encoded by codonsrepresenting both exons. Hence, the isolated polypeptides of the presentinvention comprising an amino acid sequence according to one of SEQ IDNOs:11, 88, and 89 all represent novel junction polypeptides in whichamino acid residues encoded by exon 14 are joined with amino acidresidues encoded by exon 17.

And, the isolated polypeptides of the present invention comprising anamino acid sequence according to one of SEQ ID NOs:12 and 91-93represent novel amino acid sequence fragments of the BRCA1 proteinvariant resulting from Deletion No. 6. As depicted in FIG. 9, anddescribed above, these polypeptides correspond to codon sequences inmutant mRNAs created by the direct splicing of exon 13 to exon 21—mutantmRNAs that are transcribed from mutant alleles of the BRCA1 gene bearingDeletion No. 6—and all contain certain amino acid residues encoded bycodons representing both exons. Hence, the isolated polypeptides of thepresent invention comprising an amino acid sequence according to one ofSEQ ID NOs:12 and 91-93 all represent novel junction polypeptides inwhich amino acid residues encoded by exon 13 are joined with amino acidresidues encoded by exon 21.

Further, the isolated polypeptides of the present invention comprisingan amino acid sequence according to one of SEQ ID NOs:13 and 90represent novel amino acid sequence fragments of a new carboxy-terminusadded to the BRCA1 protein variant resulting from Deletion No. 5. Asillustrated in FIG. 8, these polypeptides are encoded by codons in exon17 which are translated from a shifted reading frame created by the thesplicing of exon 14 to exon 17.

Similarly, the isolated polypeptide of the present invention comprisingan amino acid sequence according to SEQ ID NO:14 represent a novel aminoacid sequence fragments of a new carboxy-terminus added to the BRCA1protein variant resulting from Deletion No. 6. As illustrated in FIG. 9,this polypeptide is encoded by codons in exons 21, 22, 23 and 24 whichare translated from a shifted reading frame created by the the splicingof exon 13 to exon 21.

In a specific embodiment, the present invention provides isolated BRCA1protein variants having one or more amino acid sequences according toone of SEQ ID NOs:10-14 or 83-93. For example, the isolated BRCA1protein variant can be the protein variant isolated from a patienthaving Deletion No. 1, 2, 3 or 4. Alternatively, the isolated BRCA1protein variant can be the protein variant isolated from a patienthaving Deletion No. 5. Or, the isolated BRCA1 protein variant can be theprotein variant isolated from a patient having Deletion No. 6.Preferably the isolated BRCA1 protein variants contain at least 10, 20,30, 40, 50 or 60 amino acid residues which encompass a BRCA1 variantamino acid sequences provided by SEQ ID NOs:10-14 and 83-93.Additionally, the isolated BRCA1 protein variants of the presentinvention may also include other amino acid variants, such as thosecreated as a result of single nucleotide polymorphisms in the codingsequence of the BRCA1 gene.

It should be understood that hybrid proteins having one of the abovemutant BRCA1 amino acid sequences and a non-BRCA1 amino acid sequencealso fall within the scope of the present invention.

As will be apparent to a skilled artisan, the isolated nucleic acids andpolypeptides of the present invention can be prepared using techniquesgenerally known in the field of molecular biology. See generally,Sambrook et al., Molecular Cloning: A Laboratory Manual, 2^(nd) ed.,Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., 1989.

5. Antibodies

The present invention also provides antibodies selectivelyimmunoreactive with an isolated BRCA1 protein variant of the presentinvention. As used herein, the term “antibody” encompasses bothmonoclonal and polyclonal antibodies that fall within any antibodyclasses, e.g., IgG, IgM, IgA, etc. The term “antibody” also includesantibody fragments including, but not limited to, Fab and F(ab′)₂,conjugates of such fragments, and single-chain antibodies that can bemade in accordance with U.S. Pat. No. 4,704,692, which is incorporatedherein by reference. Specifically, as used herein, the phrase“selectively immunoreactive with an isolated BRCA1 protein variant ofthe present invention” means that the immunoreactivity of the antibodyof the present invention with a BRCA1 protein variant of the presentinvention is substantially higher than that with a BRCA1 proteinheretofore known in the art so that the binding of the antibody to theprotein variant of the present invention is readily distinguishable fromthe binding of the antibody to the BRCA1 protein known in the art basedon the strength of the binding affinities. Preferably, the bindingconstant differs by a magnitude of at least 2 fold, more preferably atleast 5 fold, even more preferably at least 10 fold, and most preferablyat least 100 fold.

To make the antibody, a BRCA1 protein variant of the present invention,or a suitable fragment thereof, can be used to immunize an animal. TheBRCA1 protein variant can be made by any methods known in the art, e.g.,by recombinant expression or chemical synthesis. Additionally, a mutantBRCA1 protein fragment having an amino acid sequence selected from SEQID NOs:10-14 or 83-93 can also be used. Preferably, the mutant BRCA1protein fragment consists of less than 100 amino acids, more preferablyless than 50 amino acids, and even more preferably less than 25 aminoacids. As a result, a greater portion of the total antibodies may beselectively immunoreactive with a BRCA1 protein variant of the presentinvention. Techniques for immunizing animals for the purpose of makingpolyclonal antibodies are generally known in the art. See Harlow andLane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory,Cold Spring Harbor, N.Y., 1988. A carrier may be necessary to increasethe immunogenicity of the polypeptide. Suitable carriers known in theart include, but are not limited to, liposome, macromolecular protein orpolysaccharide, or combination thereof. Preferably, the carrier has amolecular weight in the range of about 10,000 to 1,000,000. Thepolypeptide may also be administered along with an adjuvant, e.g.,complete Freund's adjuvant.

The antibodies of the present invention preferably are monoclonal. Suchmonoclonal antibodies may be developed using any conventional techniquesknown in the art. For example, the popular hybridoma method disclosed inKohler and Milstein, Nature, 256:495-497 (1975) is now a well-developedtechnique that can be used in the present invention. See U.S. Pat. No.4,376,110, which is incorporated herein by reference. Essentially,B-lymphocytes producing a polyclonal antibody against a protein variantof the present invention can be fused with myeloma cells to generate alibrary of hybridoma clones. The hybridoma population is then screenedfor antigen binding specificity and also for immunoglobulin class(isotype). In this manner, pure hybridoma clones producing specifichomogenous antibodies can be selected. See generally, Harlow and Lane,Antibodies: A Laboratory Manual, Cold Spring Harbor Press, 1988.Alternatively, other techniques known in the art may also be used toprepare monoclonal antibodies, which include but are not limited to theEBV hybridoma technique, the human N-cell hybridoma technique, and thetrioma technique.

In addition, antibodies selectively immunoreactive with a proteinvariant of the present invention may also be recombinantly produced. Forexample, cDNAs prepared by PCR amplification from activatedB-lymphocytes or hybridomas may be cloned into an expression vector toform a cDNA library, which is then introduced into a host cell forrecombinant expression. The cDNA encoding a specific desired protein maythen be isolated from the library. The isolated cDNA can be introducedinto a suitable host cell for the expression of the protein. Thus,recombinant techniques can be used to recombinantly produce specificnative antibodies, hybrid antibodies capable of simultaneous reactionwith more than one antigen, chimeric antibodies (e.g., the constant andvariable regions are derived from different sources), univalentantibodies which comprise one heavy and light chain pair coupled withthe Fc region of a third (heavy) chain, Fab proteins, and the like. SeeU.S. Pat. No. 4,816,567; European Patent Publication No. 0088994; Munro,Nature, 312:597 (1984); Morrison, Science, 229:1202 (1985); Oi et al.,BioTechniques, 4:214 (1986); and Wood et al., Nature, 314:446-449(1985), all of which are incorporated herein by reference. Antibodyfragments such as Fv fragments, single-chain Fv fragments (scFv), Fab′fragments, and F(ab′)₂ fragments can also be recombinantly produced bymethods disclosed in, e.g., U.S. Pat. No. 4,946,778; Skerra & Plückthun,Science, 240:1038-1041 (1988); Better et al., Science, 240:1041-1043(1988); and Bird, et al., Science, 242:423-426 (1988), all of which areincorporated herein by reference.

In a preferred embodiment, the antibodies provided in accordance withthe present invention are partially or fully humanized antibodies. Forthis purpose, any methods known in the art may be used. For example,partially humanized chimeric antibodies having V regions derived fromthe tumor-specific mouse monoclonal antibody, but human C regions aredisclosed in Morrison and Oi, Adv. Immunol., 44:65-92 (1989). Inaddition, fully humanized antibodies can be made using transgenicnon-human animals. For example, transgenic non-human animals such astransgenic mice can be produced in which endogenous immunoglobulin genesare suppressed or deleted, while heterologous antibodies are encodedentirely by exogenous immunoglobulin genes, preferably humanimmunoglobulin genes, recombinantly introduced into the genome. Seee.g., U.S. Pat. Nos. 5,530,101; 5,545,806; 6,075,181; PCT PublicationNo. WO 94/02602; Green et. al., Nat. Genetics, 7: 13-21 (1994); andLonberg et al., Nature 368: 856-859 (1994), all of which areincorporated herein by reference. The transgenic non-human host animalmay be immunized with suitable antigens such as a protein variant of thepresent invention to illicit specific immune response thus producinghumanized antibodies. In addition, cell lines producing specifichumanized antibodies can also be derived from the immunized transgenicnon-human animals. For example, mature B-lymphocytes obtained from atransgenic animal producing humanized antibodies can be fused to myelomacells and the resulting hybridoma clones may be selected for specifichumanized antibodies with desired binding specificities. Alternatively,cDNAs may be extracted from mature B-lymphocytes and used inestablishing a library that is subsequently screened for clones encodinghumanized antibodies with desired binding specificities. In addition,antibodies may also be produced in transgenic plants containingrecombinant nucleic acids encoding antibodies.

In accordance with another embodiment of the present invention, aprotein microchip or microarray is provided having (1) a BRCA1 proteinvariant of the present invention or a fragment thereof comprising anamino acid sequence according to SEQ ID NOs:10-14 or 83-93; and/or (2)an antibody selectively immunoreactive with a BRCA1 protein variant ofthe present invention.

Protein microarrays are becoming increasingly important in bothproteomics research and protein-based detection and diagnosis ofdiseases. The protein microarrays in accordance with the presentinvention will be useful in a variety of applications including, e.g.,high throughput screening for compounds capable of modulating theactivities of a BRCA1 protein variant of the present invention. Theprotein microarrays are also useful in detecting the mutant BRCA1proteins, and thus can be used in determining a predisposition tocancer, particularly breast cancer and ovarian cancer in patients.

The protein microarray of the present invention can be prepared by anumber of methods known in the art. An example of a suitable method isthat disclosed in MacBeath and Schreiber, Science, 289:1760-1763 (2000).Essentially, glass microscope slides are treated with analdehyde-containing silane reagent (SuperAldehyde Substrates purchasedfrom TeleChem International, Cupertino, Calif.). Nanoliter volumes ofprotein samples in a phophate-buffered saline with 40% glycerol are thenspotted onto the treated slides using a high-precision contact-printingrobot. After incubation, the slides are immersed in a bovine serumalbumin (BSA)-containing buffer to quench the unreacted aldehydes and toform a BSA layer which functions to prevent non-specific protein bindingin subsequent applications of the microchip. Alternatively, as disclosedin MacBeath and Schreiber, proteins or protein complexes of the presentinvention can be attached to a BSA-NHS slide by covalent linkages.BSA-NHS slides are fabricated by first attaching a molecular layer ofBSA to the surface of glass slides and then activating the BSA withN,N′-disuccinimidyl carbonate. As a result, the amino groups of thelysine, asparate, and glutamate residues on the BSA are activated andcan form covalent urea or amide linkages with protein samples spotted onthe slides. See MacBeath and Schreiber, Science, 289:1760-1763 (2000).

Another example of useful method for preparing the protein microchip ofthe present invention is that disclosed in PCT Publication Nos. WO00/4389A2 and WO 00/04382, both of which are assigned to Zyomyx and areincorporated herein by reference. First, a substrate or chip base iscovered with one or more layers of thin organic film to eliminate anysurface defects, insulate proteins from the base materials, and toensure a uniform protein array. Next, a plurality of protein-capturingagents (e.g., antibodies, peptides, etc.) are arrayed and attached tothe base that is covered with the thin film. Proteins or proteincomplexes can then be bound to the capturing agents forming a proteinmicroarray. The protein microchips are kept in flow chambers with anaqueous solution.

The protein microarray of the present invention can also be made by themethod disclosed in PCT Publication No. WO 99/36576 assigned to PackardBioscience Company, which is incorporated herein by reference. Forexample, a three-dimensional hydrophilic polymer matrix, i.e., a gel, isfirst deposited on a solid substrate such as a glass slide. The polymermatrix gel is capable of expanding or contracting and contains acoupling reagent that reacts with amine groups. Thus, proteins andprotein complexes can be contacted with the matrix gel in an expandedaqueous and porous state to allow reactions between the amine groups onthe protein or protein complexes with the coupling reagents thusimmobilizing the proteins and protein complexes on the substrate.Thereafter, the gel is contracted to embed the attached proteins andprotein complexes in the matrix gel.

Alternatively, the proteins and protein complexes of the presentinvention can be incorporated into a commercially available proteinmicrochip, e.g., the ProteinChip System from Ciphergen Biosystems Inc.,Palo Alto, Calif. The ProteinChip System comprises metal chips having atreated surface that interact with proteins. Basically, a metal chipsurface is coated with a silicon dioxide film. The molecules of interestsuch as proteins and protein complexes can then be attached covalentlyto the chip surface via a silane coupling agent.

The protein microchips of the present invention can also be preparedwith other methods known in the art, e.g., those disclosed in U.S. Pat.Nos. 6,087,102, 6,139,831, 6,087,103; PCT Publication Nos. WO 99/60156,WO 99/39210, WO 00/54046, WO 00/53625, WO 99/51773, WO 99/35289, WO97/42507, WO 01/01142, WO 00/63694, WO 00/61806, WO 99/61148, WO99/40434, all of which are incorporated herein by reference.

6. Genotyping

In another aspect of the present invention, methods are provided forpredicting, in an individual, the likelihood of developing cancer. Asdescribed above, the large deletions in BRCA1 genes identified inaccordance with the present invention are deleterious and predisposeindividuals having the deletions to cancer, particularly breast cancerand ovarian cancer. Thus, by detecting, in an individual, the presenceor absence of one or more of the BRCA1 variants of the presentinvention, one can reasonably predict a predisposition to cancer, e.g.,breast cancer and ovarian cancer.

Numerous techniques for detecting genetic variants are known in the artand can all be used for the method of this invention. The techniques canbe nucleic acid-based or protein-based. In either case, the techniquesused must be sufficiently sensitive so as to accurately detect thenucleotide or amino acid variations. Very often, a probe is utilizedwhich is labeled with a detectable marker. Unless otherwise specified ina particular technique described below, any suitable marker known in theart can be used, including but not limited to, radioactive isotopes,fluorescent compounds, biotin which is detectable using strepavidin,enzymes (e.g., alkaline phosphatase), substrates of an enzyme, ligandsand antibodies, etc. See Jablonski et al., Nucleic Acids Res.,14:6115-6128 (1986); Nguyen et al., Biotechniques, 13:116-123 (1992);Rigby et al., J. Mol. Biol., 113:237-251 (1977).

In a DNA-based detection method, a target DNA sample, i.e., a samplecontaining BRCA1 gene sequence should be obtained from the individual tobe tested. Any tissue or cell sample containing the BRCA1 genomic DNA ormRNA, or a portion thereof, can be used. Preferably, a tissue samplecontaining cell nuclei and thus genomic DNA can be obtained from theindividual. Blood samples can also be useful, except that only whiteblood cells and other lymphocytes have cell nuclei, while red bloodcells are enucleated and contain mRNA. Nevertheless, mRNA is also usefulas it can be analyzed for the presence of nucleotide variants in itssequence or serve as template for cDNA synthesis. The tissue or cellsamples can be analyzed directly without much processing. Alternatively,nucleic acids including the target BRCA1 nucleic acids can be extracted,purified, or amplified before they are subject to the various detectingprocedures discussed below. Other than tissue or cell samples, cDNAs orgenomic DNAs from a cDNA or genomic DNA library constructed using atissue or cell sample obtained from the individual to be tested are alsouseful.

To determine the presence or absence of the deletion mutationsidentified in the present invention, one technique is simply sequencingthe target BRCA1 genomic DNA or cDNA, particularly the region spanningthe deletion locus to be detected. Various sequencing techniques aregenerally known and widely used in the art including the Sanger methodand the Gilbert chemical method. The newly developed pyrosequencingmethod monitors DNA synthesis in real time using a luminometricdetection system. Pyrosequencing has been shown to be effective inanalyzing genetic polymorphisms such as single-nucleotide polymorphismsand can also be used in the present invention. See Nordstrom et al.,Biotechnol. Appl. Biochem., 31(2):107-112 (2000); Ahmadian et al., Anal.Biochem., 280:103-110 (2000). For example, sequencing primers can bedesigned based on either mutant or wild-type BRCA1 gene intronic orexonic sequences such that the primers have the nucleotide sequenceadjacent to a deletion locus identified in accordance with the presentinvention. In another example, PCR primers are designed based on eithermutant or wild-type BRCA1 gene intronic or exonic sequences such thatPCR amplification generates a BRCA1 DNA fragment spanning the deletionlocus. As the large deletions identified in accordance with the presentinvention alter the size of the BRCA1 genomic DNA or cDNA, the presenceor absence of a deletion mutation according to the present invention canbe determined based on the molecular weight of the PCR amplificationproducts generated using the PCR primers. Optionally, DNA sequencing isthen performed on the amplified fragment to determine the nucleotidesequence of the suspect region.

Alternatively, the restriction fragment length polymorphism (RFLP)method may also prove to be a useful technique. In particular, the largedeletions identified in accordance with the present invention result inthe elimination and creation of restriction enzyme recognition sites.Digestion of the mutant BRCA1 genomic DNAs or cDNAs with appropriaterestriction enzyme(s) will generate restriction fragment length patternsdistinct from those generated from wild-type BRCA1 genomic DNA or cDNA.Thus, the large deletions in BRCA1 of the present invention can bedetected by RFLP. The application of the RFLP techniques known in theart to the present invention will be apparent to skilled artisans.

Similarly, genomic DNA can be obtained from a patient sample anddigested by appropriate restriction enzyme(s). Southern blot can beperformed using a probe having a wild-type BRCA1 sequence that ismissing from one or more of the BRCA1 genetic variants of the presentinvention. Alternatively, probes specific to the mutant BRCA1 nucleicacids of the present invention can also be used.

The presence or absence of a BRCA1 deletion mutation identifiedaccording to the present invention can also be detected using theamplification refractory mutation system (ARMS) technique. See e.g.,European Patent No. 0,332,435; Newton et al., Nucleic Acids Res.,17:2503-2515 (1989); Fox et al., Br. J. Cancer, 77:1267-1274 (1998);Robertson et al., Eur. Respir. J., 12:477-482 (1998). In the ARMSmethod, a primer is synthesized matching the nucleotide sequenceimmediately 5′ upstream from the locus being tested except that the3′-end nucleotide which corresponds to the nucleotide at the locus is apredetermined nucleotide. For example, the 3′-end nucleotide can be thesame as that in the mutated locus. The primer can be of any suitablelength so long as it hybridizes to the target DNA under stringentconditions only when its 3′-end nucleotide matches the nucleotide at thelocus being tested. Preferably the primer has at least 12 nucleotides,more preferably from about 18 to 50 nucleotides. If the individualtested has a mutation at the locus and the nucleotide therein matchesthe 3′-end nucleotide of the primer, then the primer can be furtherextended upon hybridizing to the target DNA template, and the primer caninitiate a PCR amplification reaction in conjunction with anothersuitable PCR primer. In contrast, if the nucleotide at the locus is ofwild type, then primer extension cannot be achieved. Various forms ofARMS techniques developed in the past few years can be used. See e.g.,Gibson et al., Clin. Chem. 43:1336-1341 (1997). Thus, for example,primers having a sequence selected from SEQ ID NOs:42-47, 53-63, and70-79 can all be useful in this technique.

Similar to the ARMS technique is the mini sequencing or singlenucleotide primer extension method, which is based on the incorporationof a single nucleotide. An oligonucleotide primer matching thenucleotide sequence immediately 5′ to the locus being tested ishybridized to the target DNA or mRNA in the presence of labeleddideoxyribonucleotides. A labeled nucleotide is incorporated or linkedto the primer only when the dideoxyribonucleotides matches thenucleotide at the variant locus being detected. Thus, the identity ofthe nucleotide at the variant locus can be revealed based on thedetection label attached to the incorporated dideoxyribonucleotides. SeeSyvanen et al., Genomics, 8:684-692 (1990); Shumaker et al., Hum.Mutat., 7:346-354 (1996); Chen et al., Genome Res., 10:549-547 (2000).

Another set of techniques useful in the present invention is theso-called “oligonucleotide ligation assay” (OLA) in whichdifferentiation between a wild-type locus and a mutation is based on theability of two oligonucleotides to anneal adjacent to each other on thetarget DNA molecule allowing the two oligonucleotides joined together bya DNA ligase. See Landergren et al., Science, 241:1077-1080 (1988); Chenet al, Genome Res., 8:549-556 (1998); Iannone et al., Cytometry,39:131-140 (2000). Thus, for example, to detect a mutation at aparticular locus in the BRCA1 gene, two oligonucleotides can besynthesized, one having the BRCA1 sequence just 5′ upstream from thelocus with its 3′ end nucleotide being identical to the nucleotide inthe mutant locus of the BRCA1 gene, the other having a nucleotidesequence matching the BRCA1 sequence immediately 3′ downstream from thelocus in the BRCA1 gene. The oligonucleotides can be labeled for thepurpose of detection. Upon hybridizing to the target BRCA1 gene under astringent condition, the two oligonucleotides are subjected to ligationin the presence of a suitable ligase. The ligation of the twooligonucleotides would indicate that the target DNA has a nucleotidevariant at the locus being detected. Thus, for example, oligonucleotidescan be readily designed based on the deletion loci present in mutantBRCA1 genomic DNA or cDNA sequences that result from Deletion Nos. 1, 2,3, 4, 5, or 6.

Detection of the genetic variations identified in accordance with thepresent invention can also be accomplished by a variety ofhybridization-based approaches. Allele-specific oligonucleotides areuseful. See Conner et al., Proc. Natl. Acad. Sci. USA, 80:278-282(1983); Saiki et al, Proc. Natl. Acad. Sci. USA, 86:6230-6234 (1989).Oligonucleotide probes hybridizing specifically to a BRCA1 gene allelehaving a particular gene variant at a particular locus but not to otheralleles can be designed by methods known in the art. The probes can havea length of, e.g., from 10 to about 50 nucleotide bases. The targetBRCA1 genomic DNA or cDNA and the oligonucleotide probe can be contactedwith each other under conditions sufficiently stringent such that thegenetic variant can be distinguished from the wild-type BRCA1 gene basedon the presence or absence of hybridization. The probe can be labeled toprovide detection signals. Alternatively, the allele-specificoligonucleotide probe can be used as a PCR amplification primer in an“allele-specific PCR” and the presence or absence of a PCR product ofthe expected length would indicate the presence or absence of aparticular genetic variant. In this respect, oligos having a sequenceselected from SEQ ID NOs:7-9, 15-79 and 81 can be used.

Another useful technique that is gaining increased popularity is massspectrometry. See Graber et al., Curr. Opin. Biotechnol., 9:14-18(1998). For example, in the primer oligo base extension (PROBE™) method,a target nucleic acid is immobilized to a solid-phase support. A primeris annealed to the target immediately 5′ upstream from the locus to beanalyzed. Primer extension is carried out in the presence of a selectedmixture of deoxyribonucelotides and dideoxyribonucleotides. Theresulting mixture of newly extended primers is then analyzed byMALDI-TOF. See e.g., Monforte et al., Nat. Med., 3:360-362 (1997). Inanother example, primers can be designed based on either mutant orwild-type BRCA1 gene intronic or exonic sequences such that the primershave the nucleotide sequences adjacent to and flanking a deletion locusidentified in accordance with the present invention. PCR amplificationon a patient sample is carried out using the primers. Mass spectrometryis then performed on the PCR product.

In addition, the microchip or microarray technologies are alsoapplicable to the detection method of the present invention.Essentially, in microchips, a large number of different oligonucleotideprobes are immobilized in an array on a substrate or carrier, e.g., asilicon chip or glass slide. Target nucleic acid sequences to beanalyzed can be contacted with the immobilized oligonucleotide probes onthe microchip. See Lipshutz et al., Biotechniques, 19:442-447 (1995);Chee et al., Science, 274:610-614 (1996); Kozal et al., Nat. Med.2:753-759 (1996); Hacia et al., Nat. Genet., 14:441-447 (1996); Saiki etal., Proc. Natl. Acad. Sci. USA, 86:6230-6234 (1989); Gingeras et al.,Genome Res., 8:435-448 (1998). Alternatively, the multiple targetnucleic acid sequences to be studied are fixed onto a substrate and anarray of probes is contacted with the immobilized target sequences. SeeDrmanac et al., Nat. Biotechnol., 16:54-58 (1998). Numerous microchiptechnologies have been developed incorporating one or more of the abovedescribed techniques for detecting mutations particularly SNPs. Themicrochip technologies combined with computerized analysis tools allowfast screening in a large scale. The adaptation of the microchiptechnologies to the present invention will be apparent to a person ofskill in the art apprised of the present disclosure. See, e.g., U.S.Pat. No. 5,925,525 to Fodor et al; Wilgenbus et al., J. Mol. Med.,77:761-786 (1999); Graber et al., Curr. Opin. Biotechnol., 9:14-18(1998); Hacia et al., Nat. Genet., 14:441-447 (1996); Shoemaker et al.,Nat. Genet., 14:450-456 (1996); DeRisi et al., Nat. Genet., 14:457-460(1996); Chee et al., Nat. Genet., 14:610-614 (1996); Lockhart et al.,Nat. Genet., 14:675-680 (1996); Drobyshev et al., Gene, 188:45-52(1997).

As is apparent from the above survey of the suitable detectiontechniques, it may or may not be necessary to amplify the target DNA,i.e., the BRCA1 genomic DNA or cDNA sequence to increase the number oftarget DNA molecules, depending on the detection techniques used. Forexample, most PCR-based techniques combine the amplification of aportion of the target and the detection of mutations. PCR amplificationis well known in the art and is disclosed in U.S. Pat. Nos. 4,683,195and 4,800,159, both of which are incorporated herein by reference. Fornon-PCR-based detection techniques, if necessary, the amplification canbe achieved by, e.g., in vivo plasmid multiplication, or by purifyingthe target DNA from a large amount of tissue or cell samples. Seegenerally, Sambrook et al., Molecular Cloning: A Laboratory Manual,2^(nd) ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.,1989. However, even with scarce samples, many sensitive techniques havebeen developed in which genetic variations can be detected withouthaving to amplify the target DNA in the sample. For example, techniqueshave been developed that amplify the signal as opposed to the target DNAby, e.g., employing branched DNA or dendrimers that can hybridize to thetarget DNA. The branched or dendrimer DNAs provide multiplehybridization sites for hybridization probes to attach thereto thusamplifying the detection signals. See Detmer et al., J. Clin.Microbiol., 34:901-907 (1996); Collins et al., Nucleic Acids Res.,25:2979-2984 (1997); Horn et al., Nucleic Acids Res., 25:4835-4841(1997); Horn et al., Nucleic Acids Res., 25:4842-4849 (1997); Nilsen etal., J. Theor. Biol., 187:273-284 (1997).

A number of other techniques that avoid amplification all togetherinclude, e.g., surface-enhanced resonance Raman scattering (SERRS),fluorescence correlation spectroscopy, and single-moleculeelectrophoresis. In SERRS, a chromophore-nucleic acid conjugate isabsorbed onto colloidal silver and is irradiated with laser light at aresonant frequency of the chromophore. See Graham et al., Anal. Chem.,69:4703-4707 (1997). The fluorescence correlation spectroscopy is basedon the spatio-temporal correlations between fluctuating light signalsand trapping single molecules in an electric field. See Eigen et al.,Proc. Natl. Acad. Sci. USA, 91:5740-5747 (1994). In single-moleculeelectrophoresis, the electrophoretic velocity of a fluorescently taggednucleic acid is determined by measuring the time required for themolecule to travel a predetermined distance between two laser beams. SeeCastro et al., Anal. Chem., 67:3181-3186 (1995). Additionally, theInvader assay and the rolling circle amplification technique may also beused. See e.g. Lyamichev et al., Nat. Biotechnol., 17:292-296 (1999);Lizardi et al., Nature Genetics, 19:225-232 (1998).

In addition, the allele-specific oligonucleotides (ASO) can also be usedin in situ hybridization using tissues or cells as samples. Theoligonucleotide probes which can hybridize differentially with thewild-type gene sequence or the gene sequence harboring a mutation may belabeled with radioactive isotopes, fluorescence, or other detectablemarkers. In situ hybridization techniques are well known in the art andtheir adaptation to the present invention for detecting the presence orabsence of a genetic variant in the BRCA1 gene of a particularindividual should be apparent to a skilled artisan apprised of thisdisclosure.

Protein-based detection techniques may also prove to be useful,especially when the genetic variant causes amino acid substitutions ordeletions or insertions that affect the protein primary, secondary ortertiary structure. To detect the amino acid variations, proteinsequencing techniques may be used. For example, a BRCA1 protein orfragment thereof can be synthesized by recombinant expression using aBRCA1 DNA fragment isolated from an individual to be tested. Preferably,a BRCA1 cDNA fragment of no more than 100 to 150 base pairs encompassingthe polymorphic locus to be determined is used. The amino acid sequenceof the peptide can then be determined by conventional protein sequencingmethods. Alternatively, the recently developed HPLC-microscopy tandemmass spectrometry technique can be used for determining the amino acidsequence variations. In this technique, proteolytic digestion isperformed on a protein, and the resulting peptide mixture is separatedby reversed-phase chromatographic separation. Tandem mass spectrometryis then performed and the data collected therefrom is analyzed. SeeGatlin et al., Anal. Chem., 72:757-763 (2000).

Other useful protein-based detection techniques include immunoaffinityassays based on antibodies selectively immunoreactive with mutant BRCA1proteins according to the present invention. Such antibodies may reactspecifically with epitopes comprising the polypeptide fragments spanningthe junction regions of BRCA1 proteins that correspond to deletion lociin the mutant BRCA1 mRNAs transcribed from the mutant BRCA1 genomic DNAsof the present invention (i.e., the deletion loci of variant BRCA1polypeptides produced as a result of Deletion Nos. 1-6. Alternatively,such antibodies may react specifically with epitopes present on thenovel carboxyl-terminal polypeptides of the BRCA1 protein variantsresulting from Deletion Nos. 5 and 6. Methods for producing suchantibodies are described above in detail. Antibodies can be used toimmunoprecipitate specific proteins from solution samples or toimmunoblot proteins separated by, e.g., polyacrylamide gels.Immunocytochemical methods can also be used in detecting specificprotein polymorphisms in tissues or cells. Other well knownantibody-based techniques can also be used including, e.g.,enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA),immunoradiometric assays (IRMA) and immunoenzymatic assays (IEMA),including sandwich assays using monoclonal or polyclonal antibodies. Seee.g., U.S. Pat. Nos. 4,376,110 and 4,486,530, both of which areincorporated herein by reference.

It is noted that heterozygotes of the BRCA1 genetic variants of thepresent invention are predisposed to cancer such as breast cancer andovarian cancer. That is, as long as an individual has one chromosomecontaining a BRCA1 genetic variant of the present invention, there is anincreased likelihood of breast cancer and/or ovarian cancer in theindividual.

Thus, various techniques can be used in genotyping a BRCA1 gene of anindividual to determine, in the individual, the presence or absence of aBRCA1 genetic variant selected from the group consisting of DeletionNos. 1 to 6. Typically, once the presence or absence of a BRCA 1 geneticvariant of the present invention is determined, the result can be castin a communicable form that can be communicated to the individualpatient. Such a form can vary and can be tangible or intangible. Theresult with regard to the presence or absence of a BRCA1 genetic variantof the present invention in the individual tested can be embodied indescriptive statements, diagrams, photographs, charts, images or anyother visual forms. For example, images of gel electrophoresis of PCRproducts can be used in explaining the results. Diagrams showing where adeletion occurs in an individual's BRCA1 gene are also useful incommunicating the test results. The statements and visual forms can berecorded on a tangible media such as papers, computer readable mediasuch as floppy disks, compact disks, etc., or on an intangible media,e.g., an electronic media in the form of e-mail, or on a preferablysecured website on the internet or an intranet. In addition, the resultwith regard to the presence or absence of a BRCA1 genetic variant of thepresent invention in the individual tested can also be recorded in asound form and transmitted through any suitable media, e.g., analog ordigital cable lines, fiber optic cables, etc., via telephone, facsimile,wireless mobile phone, internet phone and the like.

The present invention also provides kits for practicing the genotypingmethods described above. The kits may include a carrier for the variouscomponents of the kit. The carrier can be a container or support, in theform of, e.g., bag, box, tube, rack, and is optionallycompartmentalized. The carrier may define an enclosed confinement forsafety purposes during shipment and storage. The kit also includesvarious components useful in detecting nucleotide or amino acid variantsdiscovered in accordance with the present invention using theabove-discussed detection techniques.

In one embodiment, the detection kit includes one or moreoligonucleotides useful in detecting the genetic variants in BRCA1 genesequence in accordance with the present invention. Preferably, theoligonucleotides are designed such that they are specific to a BRCA1nucleic acid variant of the present invention under stringentconditions. That is, the oligonucleotides should be designed such thatit can be used in distinguishing one genetic variant from another at aparticular locus under predetermined stringent hybridization conditions.Examples of such oligonucleotides include nucleic acids having asequence selected from SEQ ID NOs:7-9, 15-79 and 81. Thus, theoligonucleotides can be used in mutation-detecting techniques such asallele-specific oligonucleotides (ASO), allele-specific PCR,TaqMan-based quantitative PCR, chemiluminescence-based techniques,molecular beacons, and improvements or derivatives thereof, e.g.,microchip technologies.

In another embodiment of this invention, the kit includes one or moreoligonucleotides suitable for use in detecting techniques such as ARMS,oligonucleotide ligation assay (OLA), and the like. For example, theoligonucleotides in this embodiment include a BRCA1 gene sequenceimmediately 5′ upstream from a deletion locus to be analyzed. The 3′ endnucleotide of the oligo is the first nucleotide on the 3′ side of thedeletion locus. Examples of suitable oligos include, but are not limitedto, those consisting of a sequence selected from SEQ ID NOs:1, 3, 5, 6,42-47, 53-63, and 73-79.

The oligonucleotides in the detection kit can be labeled with anysuitable detection marker including but not limited to, radioactiveisotopes, fluorophores, biotin, enzymes (e.g., alkaline phosphatase),enzyme substrates, ligands and antibodies, etc. See Jablonski et al.,Nucleic Acids Res., 14:6115-6128 (1986); Nguyen et al., Biotechniques,13:116-123 (1992); Rigby et al., J. Mol. Biol., 113:237-251 (1977).Alternatively, the oligonucleotides included in the kit are not labeled,and instead, one or more markers are provided in the kit so that usersmay label the oligonucleotides at the time of use.

In another embodiment of the invention, the detection kit contains oneor more antibodies selectively immunoreactive with a BRCA1 proteinvariant of the present invention. Methods for producing and using suchantibodies have been described above in detail.

Various other components useful in the detection techniques may also beincluded in the detection kit of this invention. Examples of suchcomponents include, but are not limited to, DNA polymerase, reversetranscriptase, deoxyribonucleotides, dideoxyribonucleotides otherprimers suitable for the amplification of a target DNA or mRNA sequence,RNase A, mutS protein, and the like. In addition, the detection kitpreferably includes instructions on using the kit for detecting geneticvariants in BRCA1 gene sequences, particularly the genetic variants ofthe present invention.

7. Screening Assays

The present invention further provides a method for identifyingcompounds capable of modulating, preferably enhancing the activities ofa BRCA1 protein variant of the present invention. Such compounds mayprove to be useful in treating or preventing symptoms associated withdecreased BRCA1 protein activities, e.g., cancer. For this purpose, amutant BRCA1 protein or fragment thereof containing a particulardeletion in accordance with the present invention can be used in any ofa variety of drug screening techniques. Drug screening can be performedas described herein or using well known techniques, such as thosedescribed in U.S. Pat. Nos. 5,800,998 and 5,891,628, both of which areincorporated herein by reference. The candidate therapeutic compoundsmay include, but are not limited to proteins, small peptides, nucleicacids, and analogs thereof. Preferably, the compounds are small organicmolecules having a molecular weight of no greater than 10,000 dalton,more preferably less than 5,000 dalton.

In one embodiment of the present invention, the method is primarilybased on binding affinities to screen for compounds capable ofinteracting with or binding to a BRCA1 protein variant. Compounds to bescreened may be peptides or derivatives or mimetics thereof, ornon-peptide small molecules. Conveniently, commercially availablecombinatorial libraries of compounds or phage display librariesdisplaying random peptides are used.

Various screening techniques known in the art may be used in the presentinvention. The BRCA1 protein variants (drug target) can be prepared byany suitable methods, e.g., by recombinant expression and purification.The polypeptide or fragment thereof may be free in solution butpreferably is immobilized on a solid support, e.g., in a proteinmicrochip, or on a cell surface. Various techniques for immobilizingproteins on a solid support are known in the art. For example, PCTPublication WO 84/03564 discloses synthesizing a large numbers of smallpeptide test compounds on a solid substrate, such as plastic pins orother surfaces. Alternatively, purified mutant BRCA1 protein, orfragments thereof, can be coated directly onto plates such as multi-wellplates. Non-neutralizing antibodies, i.e., antibodies capable binding tothe BRCA1 protein, or fragments thereof, that do not substantiallyaffect its biological activities may also be used for immobilizing theBRCA1 protein, or fragments thereof, on a solid support.

To affect the screening, test compounds can be contacted with theimmobilized BRCA1 protein, or fragments thereof, to allow binding tooccur and complexes to form under standard binding conditions. Eitherthe drug target or test compounds are labeled with a detectable markerusing well known labeling techniques. To identify binding compounds, onemay measure the steady state or end-point formation of the drugtarget-test compound complexes, or kinetics for the formation thereof.

Alternatively, a known ligand capable of binding to the drug target canbe used in competitive binding assays. Complexes between the knownligand and the drug target can be formed and then contacted with testcompounds. The ability of a test compound to interfere with theinteraction between the drug target and the known ligand is measuredusing known techniques. One exemplary ligand is an antibody capable ofspecifically binding the drug target. Particularly, such an antibody isespecially useful for identifying peptides that share one or moreantigenic determinants of the BRCA1 protein, or fragments thereof, andpreferably antigenic determinants specific to the BRCA1 protein variantsof the present invention.

In another embodiment, a yeast two-hybrid system may be employed toscreen for proteins or small peptides capable of interacting with aBRCA1 protein variant. For example, a battery of fusion proteins eachcontaining a random small peptide fused to e.g., Gal 4 activationdomain, can be co-expressed in yeast cells with a fusion protein havingthe Gal 4 binding domain fused to a BRCA1 protein variant. In thismanner, small peptides capable of interacting with the BRCA1 proteinvariant can be identified. Alternatively, compounds can also be testedin a yeast two-hybrid system to determine their ability to inhibit theinteraction between the BRCA1 protein variant and a known protein, whichis known to interact with the BRCA1 protein or polypeptide or fragmentthereof. Again, one example of such proteins is an antibody specificallyagainst the BRCA1 protein variant. Yeast two-hybrid systems and usethereof are generally known in the art and are disclosed in, e.g.,Bartel et al., in: Cellular Interactions in Development: A PracticalApproach, Oxford University Press, pp. 153-179 (1993); Fields and Song,Nature, 340:245-246 (1989); Chevray and Nathans, Proc. Natl. Acad. Sci.USA, 89:5789-5793 (1992); Lee et al., Science, 268:836-844 (1995); andU.S. Pat. Nos. 6,057,101, 6,051,381, and 5,525,490, all of which areincorporated herein by reference.

The compounds thus identified can be further tested for activities,e.g., in stimulating the mutant BRCA1's biological activities, e.g., inDNA repair and in interacting with its known interacting partnerproteins.

Once an effective compound is identified, structural analogs or mimeticsthereof can be produced based on rational drug design with the aim ofimproving drug efficacy and stability, and reducing side effects.Methods known in the art for rational drug design can be used in thepresent invention. See, e.g., Hodgson et al., Bio/Technology, 9:19-21(1991); U.S. Pat. Nos. 5,800,998 and 5,891,628, all of which areincorporated herein by reference. An example of rational drug design isthe development of HIV protease inhibitors. See Erickson et al.,Science, 249:527-533 (1990). Preferably, rational drug design is basedon one or more compounds selectively binding to a mutant BRCA1 proteinor a fragment thereof.

In one embodiment, the three-dimensional structure of, e.g., a BRCA1protein variant, is determined by biophysical techniques such as X-raycrystallography, computer modeling, or both. Desirably, the structure ofthe complex between an effective compound and the mutant BRCA1 proteinis determined, and the structural relationship between the compound andthe protein is elucidated. In this manner, the moieties and thethree-dimensional structure of the selected compound, i.e., leadcompound, critical to the its binding to the mutant BRCA1 protein arerevealed. Medicinal chemists can then design analog compounds havingsimilar moieties and structures. In addition, the three-dimensionalstructure of wild-type BRCA1 protein is also desirably deciphered andcompared to that of a mutant BRCA1 protein. This will aid in designingcompounds selectively interacting with the mutant BRCA1 protein.

In another approach, a selected peptide compound capable of binding theBRCA1 protein variant can be analyzed by alanine scanning mutagenesis.See Wells, et al., Methods Enzymol., 202:301-306 (1991). In thistechnique, an amino acid residue of the peptide is replaced by Alanine,and its effect on the peptide's binding affinity to the mutant BRCA1protein is tested. Amino acid residues of the selected peptide areanalyzed in this manner to determine the domains or residues of thepeptide important to its binding to mutant BRCA1 protein. These residuesor domains constituting the active region of the compound are known asits “pharmacophore”. This information can be very helpful in rationallydesigning improved compounds.

Once the pharmacophore has been elucidated, a structural model can beestablished by a modeling process which may include analyzing thephysical properties of the pharmacophore such as stereochemistry,charge, bonding, and size using data from a range of sources, e.g., NMRanalysis, x-ray diffraction data, alanine scanning, and spectroscopictechniques and the like. Various techniques including computationalanalysis, similarity mapping and the like can all be used in thismodeling process. See e.g., Perry et al., in OSAR: QuantitativeStructure-Activity Relationships in Drug Design, pp. 189-193, Alan R.Liss, Inc., 1989; Rotivinen et al., Acta Pharmaceutical Fennica,97:159-166 (1988); Lewis et al., Proc. R. Soc. Lond., 236:125-140(1989); McKinaly et al., Annu. Rev. Pharmacol. Toxiciol., 29:111-122(1989). Commercial molecular modeling systems available from PolygenCorporation, Waltham, Mass., include the CHARMm program, which performsthe energy minimization and molecular dynamics functions, and QUANTAprogram which performs the construction, graphic modeling and analysisof molecular structure. Such programs allow interactive construction,visualization and modification of molecules. Other computer modelingprograms are also available from BioDesign, Inc. (Pasadena, Calif.),Hypercube, Inc. (Cambridge, Ontario), and Allelix, Inc. (Mississauga,Ontario, Canada).

A template can be formed based on the established model. Variouscompounds can then be designed by linking various chemical groups ormoieties to the template. Various moieties of the template can also bereplaced. In addition, in the case of a peptide lead compound, thepeptide or mimetics thereof can be cyclized, e.g., by linking theN-terminus and C-terminus together, to increase its stability. Theserationally designed compounds are further tested. In this manner,pharmacologically acceptable and stable compounds with improved efficacyand reduced side effect can be developed.

8. Cell and Animal Models

In yet another aspect of the present invention, a cell line and atransgenic animal carrying a BRCA1 nucleic acid variant in accordancewith the present invention are provided. The cell line and transgenicanimal can be used as model systems for studying cancers and testingvarious therapeutic approaches in treating cancers, e.g., breast cancerand ovarian cancer.

To establish the cell line, cells expressing the mutant BRCA1 proteincan be isolated from an individual carrying the genetic variants. Theprimary cells can be transformed or immortalized using techniques knownin the art. Alternatively, normal cells expressing a wild-type BRCA1protein or other type of genetic variants can be manipulated to replacethe entire endogenous BRCA1 gene with a BRCA1 nucleic acid variant ofthe present invention, or simply to introduce mutations into theendogenous BRCA1 gene. The genetically engineered cells can further beimmortalized.

A more valuable model system is a transgenic animal. A transgenic animalcan be made by replacing its endogenous BRCA1 gene ortholog with a humanBRCA1 nucleic acid variant of the present invention. Alternatively,deletions can be introduced into the endogenous animal BRCA1 geneortholog to simulate the BRCA1 alleles discovered in accordance with thepresent invention. Techniques for making such transgenic animals arewell known and are described in, e.g., Capecchi, et al., Science,244:1288 (1989); Hasty et al., Nature, 350:243 (1991); Shinkai et al.,Cell, 68:855 (1992); Mombaerts et al., Cell, 68:869 (1992); Philpott etal., Science, 256:1448 (1992); Snouwaert et al., Science, 257:1083(1992); Donehower et al., Nature, 356:215 (1992); Hogan et al.,Manipulating the Mouse Embryo; A Laboratory Manual, 2^(nd) edition, ColdSpring Harbor Laboratory Press, 1994; and U.S. Pat. Nos. 5,800,998,5,891,628, and 4,873,191, all of which are incorporated herein byreference.

The cell line and transgenic animal are valuable tools for studying themutant BRCA1 genes, and in particular for testing in vivo the compoundsidentified in the screening method of this invention and othertherapeutic approaches as discussed above. As is well known in the art,studying drug candidates in a suitable animal model before advancingthem into human clinical trials is particularly important because notonly can efficacy of the drug candidates can be confirmed in the modelanimal, but the toxicology profiles, side effects, and dosage ranges canalso be determined. Such information is then used to guide humanclinical trials.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, it will be obvious that certain changes and modificationsmay be practiced within the scope of the appended claims.

All publications mentioned in the specification are indicative of thelevel of those skilled in the art to which this invention pertains. Allpublications and patent applications are herein incorporated byreference to the same extent as if each individual publication or patentapplication was specifically and individually indicated to beincorporated by reference.

1. An isolated nucleic acid comprising SEQ ID NO:6, and the complementthereof.
 2. The isolated nucleic acid of claim 1, wherein said isolatednucleic acid comprises SEQ ID NO:37, or the complement thereof.
 3. Amethod of making the isolated nucleic acid of claim 2 comprisingamplifying genomic DNA isolated from a sample obtained from a humanpatient.
 4. A method of making the isolated nucleic acid of claim 1comprising amplifying genomic DNA isolated from a sample obtained from ahuman patient.
 5. The method of claim 4, wherein said sample is a bloodsample.
 6. The method of claim 4, wherein said amplification is by thepolymerase chain reaction.
 7. The method of claim 4, wherein saidpatient is being evaluated for an enhanced risk of cancer.
 8. The methodof claim 4, wherein said cancer is breast or ovarian cancer.
 9. Theisolated nucleic acid of claim 1, wherein said isolated nucleic acidcomprises SEQ ID NO:38, or the complement thereof.
 10. The isolatednucleic acid of claim 2, wherein said isolated nucleic acid is a BRCA1nucleic acid comprising SEQ ID NO:37.
 11. A method of making theisolated nucleic acid of claim 9 comprising amplifying genomic DNAisolated from a sample obtained from a human patient.
 12. The isolatednucleic acid of claim 1, wherein said isolated nucleic acid comprisesSEQ ID NO:39, or the complement thereof.
 13. The isolated nucleic acidof claim 9, wherein said isolated nucleic acid is a BRCA1 nucleic acidcomprising SEQ ID NO:38.
 14. A method of making the isolated nucleicacid of claim 12 comprising amplifying genomic DNA isolated from asample obtained from a human patient.
 15. The isolated nucleic acid ofclaim 1, wherein said isolated nucleic acid comprises SEQ ID NO:40, orthe complement thereof.
 16. The isolated nucleic acid of claim 12,wherein said isolated nucleic acid is a BRCA1 nucleic acid comprisingSEQ ID NO:39.
 17. A method of making the isolated nucleic acid of claim15 comprising amplifying genomic DNA isolated from a sample obtainedfrom a human patient.
 18. The isolated nucleic acid of claim 1, whereinsaid isolated nucleic acid comprises SEQ ID NO:41, or the complementthereof.
 19. A method of making the isolated nucleic acid of claim 18comprising amplifying genomic DNA isolated from a sample obtained from ahuman patient.
 20. The isolated nucleic acid of claim 1, wherein saidisolated nucleic acid is a BRCA1 nucleic acid comprising SEQ ID NO:6.